LOCUS Exported 7709 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION All-in-one doxycycline inducible lentiviral vector for expression of one or two genes using the P2A self-cleaving peptide.. ACCESSION . VERSION . KEYWORDS pCW57-MCS1-2A-MCS2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7709) AUTHORS Barger CJ, Branick C, Chee L, Karpf AR TITLE Pan-Cancer Analyses Reveal Genomic Features of FOXM1 Overexpression in Cancer. JOURNAL Cancers (Basel). 2019 Feb 21;11(2). pii: cancers11020251. doi: 10.3390/cancers11020251. PUBMED 30795624 REFERENCE 2 (bases 1 to 7709) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7709 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 41..158 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 212..526 /label=tight TRE promoter /note="Tet-responsive promoter PTight, consisting of seven tet operator sequences followed by the minimal CMV promoter" protein_bind 220..238 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 256..274 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 291..309 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 327..345 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 363..381 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 398..416 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 434..452 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 499..523 /label=LNCX /note="Human CMV promoter, forward primer" CDS 567..623 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" promoter 651..1161 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 1080..1098 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 1171..1767 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(1171..1190) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 1667..1687 /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS 1768..1821 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 1822..2568 /codon_start=1 /product="improved tetracycline-controlled transactivator" /label=rtTA-Advanced /note="In the Tet-On(R) system, rtTA-Advanced binds to the Tet-responsive element and stimulates transcription only in the presence of tetracycline or doxycycline." /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA DALDDFDLDMLPADALDDFDLDMLPG" primer_bind complement(2608..2628) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3026..3037) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 3242..3475 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 3547..3668 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3584..3603) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3638..3657 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 3708..3843 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3770..3789 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 3843..3914 /gene="bla" /label=AmpR promoter CDS 3915..4775 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(4133..4152) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4946..5534 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5435..5454 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5688..5705 /label=L4440 /note="L4440 vector, forward primer" protein_bind 5822..5843 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 5858..5888 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5896..5912 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 5901..5923 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 5920..5936 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5920..5936 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 5955..5975 /label=T3 /note="T3 promoter, forward primer" promoter 5957..5975 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 6003..6229 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 6230..6410 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 6457..6582 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 7075..7308 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 7493..7537 /codon_start=1 /product="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" /label=gp41 peptide /note="recognized by the 2H10 single-chain llama nanobody" /translation="KNEQELLELDKWASL" ORIGIN 1 aattctcgac ctcgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga 61 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta 121 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca 181 gagatccact ttggccgcga atcgatatgt cgagtttact ccctatcagt gatagagaac 241 gtatgtcgag tttactccct atcagtgata gagaacgatg tcgagtttac tccctatcag 301 tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt 361 actccctatc agtgatagag aacgtatgtc gagtttatcc ctatcagtga tagagaacgt 421 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgaggtaggc gtgtacggtg 481 ggaggcctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gaattggcta 541 gcgaattcgt cgacaccggt tccggagcca cgaacttctc tctgttaaag caagcaggag 601 acgtggaaga aaaccccggt cctaggctgc agacgcgttg tacaggatcc ggggttgggg 661 ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc tgggcgtggt 721 tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc 781 gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc tgctccgccc 841 ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca 901 cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc gcgccgaccg 961 cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag cggccgggaa 1021 ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg 1081 tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct cgttgaccga 1141 atcaccgacc tctctcccca gcaattcacc atgaccgagt acaagcccac ggtgcgcctc 1201 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac 1261 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa 1321 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc 1381 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag 1441 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa 1501 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc 1561 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg 1621 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc 1681 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc 1741 tggtgcatga cccgcaagcc cggtgccgaa ggtagaggtt ctctcctcac ttgtggtgat 1801 gttgaagaaa accctggtcc aatgtctaga ctggacaaga gcaaagtcat aaacggagct 1861 ctggaattac tcaatggtgt cggtatcgaa ggcctgacga caaggaaact cgctcaaaag 1921 ctgggagttg agcagcctac cctgtactgg cacgtgaaga acaagcgggc cctgctcgat 1981 gccctgccaa tcgagatgct ggacaggcat catacccact tctgccccct ggaaggcgag 2041 tcatggcaag actttctgcg gaacaacgcc aagtcatacc gctgtgctct cctctcacat 2101 cgcgacgggg ctaaagtgca tctcggcacc cgcccaacag agaaacagta cgaaaccctg 2161 gaaaatcagc tcgcgttcct gtgtcagcaa ggcttctccc tggagaacgc actgtacgct 2221 ctgtccgccg tgggccactt tacactgggc tgcgtattgg aggaacagga gcatcaagta 2281 gcaaaagagg aaagagagac acctaccacc gattctatgc ccccacttct gagacaagca 2341 attgagctgt tcgaccggca gggagccgaa cctgccttcc ttttcggcct ggaactaatc 2401 atatgtggcc tggagaaaca gctaaagtgc gaaagcggcg ggccgaccga cgcccttgac 2461 gattttgact tagacatgct cccagccgat gcccttgacg actttgacct tgatatgctg 2521 cctgctgacg ctcttgacga ttttgacctt gacatgctcc ccgggtaact aagtaaggat 2581 cgatccaaga tatcgtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc 2641 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta 2701 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt 2761 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca 2821 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc 2881 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt 2941 gtcggggaag ctgacgtcct ttccatggct gctcgcctgt gttgccacct ggattctgcg 3001 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg 3061 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat 3121 ctccctttgg gccgcctccc cgcctgtttc gcctcggcgt ccggactaga ggtaccttta 3181 agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga 3241 ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct 3301 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 3361 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 3421 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtagt 3481 agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt 3541 gagaggaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 3601 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 3661 gtatcttatc atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa 3721 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag 3781 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag 3841 gcctttcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 3901 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 3961 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 4021 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 4081 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 4141 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 4201 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 4261 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 4321 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 4381 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 4441 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 4501 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 4561 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 4621 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 4681 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 4741 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 4801 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 4861 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 4921 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 4981 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 5041 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 5101 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 5161 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 5221 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 5281 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 5341 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 5401 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 5461 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 5521 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 5581 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 5641 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 5701 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 5761 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 5821 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 5881 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg 5941 attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctgcaagctt 6001 aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 6061 tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 6121 tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 6181 gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg 6241 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 6301 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 6361 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg 6421 aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt 6481 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 6541 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga 6601 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta 6661 aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta 6721 gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga 6781 tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg 6841 atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 6901 aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga 6961 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc 7021 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc 7081 tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 7141 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 7201 ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 7261 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg 7321 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa 7381 atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa 7441 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 7501 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 7561 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 7621 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 7681 tcagacccac ctcccaaccc cgaggggac //